Fig 7.

Recovery of phosphorylated lipid A species and phosphorylated dihydroceramide (PDHC) lipids in organic solvent extracts from calculus-contaminated teeth. Teeth extracted from two patients were individually pooled and extracted for lipids, as described in Materials and Methods. A sample of each lipid extract was evaluated for phosphorylated lipid A species using MRM-MS quantification of transitions characteristic for the dominant phosphorylated lipid A species detected in Fig. 5 (m/z 1,404 and 1,390 precursor ions were evaluated). (Left) Shown are the MRM-MS profiles for the m/z 1,390 to 726 (A), 1,390 to 565 (B), 1,404 to 726 (C), and 1,404 to 565 (D) ion transitions. The MS/MS transitions characteristic for the m/z 1,418 lipid A precursor ion were also evaluated and confirmed negligible levels of this lipid A in lipid extracts from diseased teeth (data not shown). The tooth lipid extracts (5 μg each) were injected at 1 and 3 min. A sample of P. gingivalis lipid A was injected at 4.5 min. In a separate analysis, we evaluated the same tooth lipid extracts for characteristic phosphorylated dihydroceramide lipids using MRM-MS analysis and previously characterized mass spectrometric parameters for these lipid products. Tooth lipid samples (5 μg each) were injected at 1 and 3.5 min. The phosphorylated dihydroceramide lipids elute in the following order from top to bottom: low-mass PE DHC lipid (m/z 677 to 140 negative-ion transition), high-mass PE DHC lipid (m/z 705 to 140 negative-ion transition), low-mass PG DHC lipid (m/z 932 to 171 negative-ion mass), high-mass PG DHC lipid (m/z 960 to 171 negative-ion mass) (31, 33). Note that the ion abundances for phosphorylated dihydroceramides are 3 or 4 orders of magnitude greater than the MS scan abundances for lipid A species.